GABA-A receptor (GAR) mediated presynaptic inhibition of afferent input to the spinal cord is critical for the establishment of nociceptive threshold. In the absence of injury, spinal administration of GAR antagonists produce hyperalgesia and allodynia whereas GAR agonists are analgesic. We recently demonstrated, however, that in the presence of persistent inflammation, there is a shift in GAR signaling such that activation of spinal GARs actually contributes to inflammatory hyperalgesia: spinal administration GAR antagonists reverse inflammatory hyperalgesia while GAR agonists exacerbate it. Our preliminary data suggest this shift reflects a presynaptic change in GAR signaling which includes the emergence of distinct GAR receptor subunits and a depolarizing shift in the anion equilibrium potential (Eanion). A depolarizing shift in Eanion may enable GAR activation to become excitatory. However, our recent data also indicate that midazolam, a benzodiazepine receptor agonist, retains analgesic efficacy in the presence of inflammation, indicating that a depolarizing shift in Eanion alone is insufficient to account for the inflammation-induced shift in GAR signaling. The apparent shift in spinal GAR signaling has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, we have proposed a series of experiments described under 4 specific aims, designed to identify mechanisms underlying spinal GAR signaling in the presence and absence of inflammation. In Specific Aim 1, we will characterize inflammation-induced changes in the biophysical properties and pharmacology of GAR mediated currents and the regulation of anion homeostasis in cutaneous sensory neurons. In Specific Aim 2, we will characterize inflammation-induced changes in the expression and distribution of GAR subunits in cutaneous sensory neurons. In Specific Aim 3, we will assess the functional consequences of inflammation-induced changes in GAR subunit expression and intracellular anion homeostasis. And in Specific Aim 4, we will determine the extent to which specific GAR subunits in cutaneous sensory neurons mediate the emergence of pronociceptive actions of GAR signaling in the presence of inflammation. The experiments described under these 4 aims, involving an array of approaches ranging from behavioral pharmacology to viral vector mediated manipulation of gene expression, are designed to test the central hypothesis that persistent inflammation results in changes in GAR signaling that reflect a combination of changes in Eanion and GAR subunit expression in primary afferent neurons. PUBLIC HEALTH RELEVANCE: 3-aminobutyric acid-A (GABA-A) receptors on the central terminals of primary afferent neurons appear to play critical role both in the inhibition of acute pain and the maintenance of persistent pain in the presence of injury. The mechanisms underlying this shift in GABA-A receptor signaling remain to be identified, but it has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, with the ultimate goals of both minimizing deleterious consequences of general anesthetics and identifying novel targets for therapeutic interventions, we have proposed a series of experiments designed to identify mechanisms underlying the shift in spinal GABA-A receptor signaling in the presence of inflammation.